Tyrosine phosphorylation is a key regulatory and signaling mechanism utilized by cells.1 In cells, tyrosine phosphorylation is dynamic, with phosphorylation by protein tyrosine kinases (PTKs) and dephosphorylation by protein tyrosine phosphatases (PTPs) occurring in concert under exquisite cellular control.2 Misregulated tyrosine phosphorylation of key signaling molecules is involved in a number of disease states, including autoimnunity and cancer.3,4 For example, one vital biological process in which tyrosine phosphorylation is crucial is T cell receptor (TCR) signaling. T cell function is both positively and negatively regulated by reversible tyrosine phosphorylation. One of the first steps following binding of an antigen to the TCR is the activation of Lck, a Src family PTK.5 Lck is positively regulated through phosphorylation of Y394 and negatively regulated through phosphorylation of Y505.5,6 Relatively minor changes in the PTP/PTK balance can have a major impact in TCR signaling and result in immunological diseases including autoimmunity, allergies and immunodeficiency.6 Although it is clear that tyrosine phosphorylation is crucial in regulating cellular signaling and that PTPs and PTKs are key targets in the treatment of many human diseases, researchers have not yet assembled the toolbox necessary to thoroughly characterize and fully assess the regulation of these enzymes in single cells. Indeed, it has been recently recognized that one of the major challenges in the field of phosphorylation-dependent signal transduction is the lack of techniques capable of dynamic and single-cell analysis of intracellular phosphorylation and dephosphorylation.7 
Despite the importance of reversible tyrosine phosphorylation in many cellular processes, techniques for detecting and measuring tyrosine phosphorylation have been relatively slow to develop. Currently, a number of phospho-tyrosine analogs are available for in vitro phosphatase assays, including small, non-peptidic substrates such as para-nitrophenylphosphate (p-NPP), 4-methylumbelliferone phosphate (MUP) and its fluorinated derivative difluoromethylumbelliferone phosphate (DiFMUP).8 For some time, intracellular protein tyrosine phosphorylation was exclusively detected using radio-labeling methods.9 More recently the development of anti-phospho-tyrosine10 and of specific anti-phospho-residue11 antibodies allowed for the development of western blotting techniques. These antibodies are also used in microscopy to characterize the spatial distribution of tyrosine phosphorylation inside cells.12 Despite the advancement in techniques, the field still suffers from lack of detection methods capable of dynamic and single-cell level analysis of tyrosine phosphorylation. Similar methods are already available for other signaling phenomena. For example, real-time single-cell analysis of intracellular calcium waves is now possible both in flow cytometry and microscopy using special calcium-sensitive fluorescent probes.13 Although PTPs are promising drug targets, the lack of adequate methods for monitoring their intracellular activity has limited the development of cell-based assays for screening of PTP inhibitors. A common challenge of developing anti-PTP small molecule inhibitors for therapy of human diseases has been so far the low cell-permeability of inhibitor lead compounds. A cell-based assay of PTP activity would help selecting cell-permeable enzyme inhibitors, and would be particularly helpful in speeding up development of novel anti-PTP therapies for a variety of human diseases.